Cytochrome P450 enzyme complexes and methods of treatment using the same

ABSTRACT

The present invention provides methods and compositions for balancing electron reduction potentials of formulations in a manner that reduces susceptibility to changes from xenobiotics. The present invention also provides novel compositions of matter based on structuring from a mobile nucleotide integral to its architecture.

This application is a continuation of U.S. patent application Ser. No.16/666,692 filed Oct. 29, 2019, which is a continuation of U.S. patentapplication Ser. No. 15/677,381 filed Aug. 15, 2017 (now U.S. Pat. No.10,493,090 issued Dec. 3, 2019), which is a continuation of U.S. patentapplication Ser. No. 13/748,990 filed Jan. 24, 2013, which is adivisional of U.S. patent application Ser. No. 12/653,052 filed Dec. 8,2009, which is a divisional of U.S. patent application Ser. No.11/312,056 filed Dec. 20, 2005, which claims priority of provisionalapplication Ser. No. 60/638,918, filed Dec. 23, 2004, the disclosures ofwhich are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

The present invention relates generally to methods and compositions fortreating living organisms.

Upon utilization of a nanoscalar particle to achieve a practicalpurpose, its design and replication defines the field of nanotechnology.Indeed, over the past decade, inventions of biomaterials have focused onsynthesis, while purposeful in vivo applications have gone wanting.Developments have resulted from the fabrication of novel architecturesthat range from Bucky Balls to octahedral DNA. Cytochromes offer somespecial nanometric advantages that are realized as a result ofarchitecture elucidated within the current invention

A diversity of cytochromes present possibilities for multitudes ofcombinations. For example, linkage of chemical reduction of dioxygen toelectrogenic translocation of protons across a membrane occurs throughCytochrome C (Cc) oxidase (A. Namslauer, A. S. Pawate, R. B. Gennis andP. Brzezinski (2003) Redox-coupled proton translocation in biologicalsystems: Proton shuttling in cytochrome c oxidase PNAS100(26):15543-15547) and if Cc, for instance, is singled out from allother cytochromes for consideration, over three score evolutionary linesare documented, a diversity that offers many avenues of process.

Cytochromes P450 (P450) are hemoproteins that catalyze monooxygenationof endogenous and xenobiotic hydrophobic substrates. Families of P450have a broad range of substrates and are responsible for processingmetabolic quantities of exogenous compounds by inserting singlet oxygenthat renders them soluble for removal. The utilization of oxygen by P450mediates hydroxylations, epoxidations, dehalogenations, deaminations anddealkylations. The predominance of literature is devoted to P450monoxygenases (CYP); however, electron reduction must, necessarily, bebalanced by coupling reactions with NADPH:Cytochrome P450 reductases(CPR). P450 is understood as an enzyme complex of CYP and CPR.

The present invention generally relates to cytochromes, particularlycytochromes P450. Specifically, the invention is of primary applicationfor electron reduction potential mechanisms that activate the induction,building, and accurate architectural reproduction by over fiftyfunctionally different structures of P450 Each monooxygenase issubstrate specific and, coupled with CPR, may be readspectrophotometrically at 450 nm in the presence of carbon monoxide and,for example, nitrobenzoate.

In particular, the present inventor has determined that small quantitiesof xenobiotics, such as carcinogens and drugs, can be recognized,deactivated and prepared for removal by P450 systems. Although, somesubstrates are beneficial, others are toxins, mutagens, or carcinogens.

It is in the best interest of humanity to maintain P450 systems atoptimal levels to metabolize and remove physiological concentrations ofobjectionable substances. The majority of xenobiotics, being unavoidablethrough environmental, gustatory, and pharmaceutical exposures, may beprocessed at some point of metabolism by P450. On the other hand,inhibitors of P450 are also unavoidable. Upon exposure to the strongestinhibitors, the population is left without the ability to defendadequately against xenobiotics. Until the discoveries of the presentinvention, a means of re-establishing and/or fortifying P450 to processxenobiotics in living animals had not been made available.

SUMMARY OF THE INVENTION

This invention elucidates a design philosophy that when applied tocytochromes, deals with physiologically important xenobioticseffectively. In certain embodiments, building and increasing quantitiesof most of the known P450 structures is accomplished in vivo as aconsequence of cascading responses to architectural modification,primarily, with the nucleotide, flavin mononucleotide (FMN), within CPR.As these nucleotides are administered into living systems, they moveacross the surface of reductases as they become integral to thestructure and function of entire monooxygenase-reductase complexes,primarily as P450 plus the added FMN (P450:FMN) (or other suitableoxidant).

Thus, a P450:FMN enzyme complex, with more than one FMN molecule in thecomplex, results in a novel composition of matter (such as in the formof a food supplement, a dietary supplement, a beverage, a topicalformulation, or a pill) that assists the body in detoxifyingxenobiotics. The direct connection of FMN, for example, to fortify theseenzyme complexes, has not been made previously. This invention thusprovides methods of detoxification by administering compositions thatare selected for accelerating specific activity. These formulationsenhance P450 in subcellular nanoarrays. By means of compositionscomprised of inducers that are selected for regeneration, the presentinvention introduces methods of activating repressed nanoscopicstructures.

For these reasons, it would be desirable to provide novel methods andcompositions for the purpose of fortifying the complexes in humans andother living organisms. It would be particularly desirable if suchmethods and compositions were able to induce these complexes in specifictissues. The present invention further provides convenient methodsresulting in fortification of these complexes for animal and humantherapy. It is desirable that the methods and compositions of thepresent invention promote eradication of undesirable compounds in amanner that enhances the quality of life. It would be further desirableto provide palatable treatments that are nutritious and broadlyeffective. To that end, preferably, the compositions of an embodiment ofthe present invention are sweet-tasting, as a rewarding means thatencourage daily upkeep of the enzymes complexes.

As a first aspect, the present invention provides isolated P450:FMNcompositions of matter wherein there is a plurality of FMN molecules inthe enzymatic complex. The methods of isolation, such as from urine orthe liver of an animal, are well known to those skilled in the art.

As a second aspect, the present invention provides a method forenhancing P450 family members, numbering over 50, by treatment withtheir corresponding oxidants. The method comprises administering to ananimal in need thereof oxidants that increase the quantity of thecomplex in that animal.

As a third aspect, the present invention provides compositions forinducing P450:FMN enzyme complexes.

As a fourth aspect, the present invention provides compositions forenhancing anticarcinogenic enzyme complexes in humans. The compositionscomprise an effective amount of a nucleotide that increases the amountof the complexes in vivo.

As a fifth aspect, the present invention provides compositionscomprising a first compound selected from the group consisting of CPRand oxidants that induce P450:FMN enzyme complexes.

As a sixth aspect, the present invention provides methods for theautomatic fortification of P450:FMN enzyme complexes in living systems.The methods include treatment with a compound selected from the groupconsisting of one or more structural components of the enzyme complexes.

As a seventh aspect, the present invention provides a method forimproving the chemical defense of humans and animals using P450:FMNenzyme complexes. The method includes the step of administering to ahuman or animal in need thereof an effective amount of a compositioncomprising FMN.

As another specific aspect, the present invention provides methods fordietary supplements to come to the defense of humans against harmfulxenobiotics whilst improving nutrition, particularly, in sweeteners.

Compositions are provided that comprise naturally sweet reductants withnatural oxidants for increasing the amount of P450:FMN enzyme complexesin an animal.

These and other aspects of the present invention are described furtherin the detailed description and examples of the invention which follow.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a simplified and integrated schematic depiction of the enzymecomplex of the invention. At the top center of the FIGURE issupplemental FMN, that is processed and integrated into native P450yielding P450:FMN of the present invention. Products are given at thebottom center of the FIGURE. Fortification of the complex is achieved byadministration of effective therapeutic dosages of one or morecomponents of the complex.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, nanoscalar methods, compositions,and systems are provided that induce P450. Methods are provided fortreatment, particularly for those who are compromised by exposure thatrequires therapy by administration of the compositions of the presentinvention.

The subject invention provides tools and methodologies for activatingproduction of structures from nucleotides, enzymes and their substrates.Of particular note, a number of nucleotides, most often as several FMNs,occur as mobile components of the enzyme complex. The subject inventionrepresents a significant contribution to fields of nutrition,nutraceuticals, dietary supplements, pharmaceuticals, cosmetics,prophylaxis, and medicine. Acceleration by formulation of nutritiveoxidants with reductants contributes to distribution.

Accordingly, the present invention provides methods for treatinganimals, for increasing the amount of cytochrome P450 in animals, andfor enhancing the ability of an animal to detoxify xenobiotics. Thepresent invention also provides compositions and formulations foradministration to animals in need thereof, or as a prophylaxis, in orderto increase the amount of cytochrome P450 in the body of the animal. Anexemplary nutritive formulation includes a selection of a slow-releaseniacin with a palatable reductant, preferably a self-rewardingsweetener, such as glucose.

Unless otherwise defined, all technical and scientific terms employedherein have their conventional meaning in the art. As used herein, thefollowing terms have the meanings ascribed to them.

Enzymatic complexes as used herein refers to the complete and functionalP450:FMN quaternary structure, wherein, the preferred molecular complexis modified with a plurality of FMN molecules. In accordance with thepresent invention, these complexes are fortified, or strengthened, in amanner that promotes, increases and improves rates of reaction of P450s.

“Oxidant” refers to an electron acceptor of CPR.

“Reductant” refers to electron donors or oxidase substrates that induceCYP. Oxidase substrates, which induce CYP accelerate the metabolism ofoxidants by CPR.

“Inducer” refers to oxidants and reductants that improve the metaboliccapacity of P450s. Induction involves a given substrate, enhancing thespecific activity of the P450s.

“P450:FMN” refers to a P450 enzyme complex with more than one FMN. “Cc”refers to Cytochrome C.

“Animal” refers to virtually all living biota of the kingdom of animalscapable of receiving treatments, particularly humans, their pets, andlivestock.

“Live organism” refers to all living organisms.

“Percent” or “%” is percent weight.

“ppm” refers to parts per million.

“g” refers to grams.

“mg” refers to milligrams.

“mcg” refers to micrograms.

“Sweetener” includes, but is not limited to, the following: naturalsugars, for example, pentose, hexose, disaccharide, and oligosaccharide;and artificial sweeteners, for example, sucralose, saccharin, andaspartame. Natural sugars may further include: xylose, arabinose,fructose, glucose, mannose, galactose, sucrose, maltose, xylitol, andthe like. The terms sweeteners may also include mixtures of one or morenatural sugars and/or one or more artificial sweeteners.

The compositions and methods of the present invention may beadministered by any therapeutically effective route of administration,including but not limited to oral, sublingual, rectal, parental(subcutaneous, intramuscular, intravenous), transdermal, topical, nasal,aerosol and like forms of administration. Dosage forms include tablets,troches, suspensions, solutions, capsules, powders, microencapsulatedsystems, various transdermal delivery systems, and the like. Topical andoral administrations are particularly preferred.

In particular, they may be advantageously administered to humans,animals, and protistans, alike because of the universality ofcytochromes P450. The methods and compositions of the preferredembodiment of the present invention may be used to enhance defensemechanisms of xenobiotics in humans and their domesticated animals.Where metabolism of medicines may be affected by prior exposure to P450inhibitors, the methods and compositions of the present invention may beused, furthermore, to assist with determination of baselines for dosageof pharmaceuticals that may be metabolized by P450.

As provided herein, fortification focuses on modulating electrontransfer through the enzymatic complexes in a manner that shifts theflow of electrons. FIG. 1 is a schematic depiction of the electrontransfer to the catalytic cycle of a single complex. By accelerating oradding to the CPR of an organ or tissue, reductive capacity is enhanced.An enhanced pool allows increased capacity for electron transfer.Accordingly, the compositions and methods of the present invention mayinclude inducers or oxidants as integral structural components.

Methods and Compositions

The present invention provides methods for administering substrates thatare designed to modify the architecture of P450s and fortify P45:FMNcomplexes in animals, including humans. These methods typically involveadministration of an oxidant component and may, preferably, includeadministration of an oxidant that is a component of the complex.Oxidants may be formulated with appropriate reductants for balancedtherapy and co-induction of monooxygenases and reductases.

Oxidants

Suitable oxidants are compounds that induce NADPH:cytochrome P450reductase. Any compound capable of inducing such reductases will beuseful as the oxidant component in the methods, compositions, andsystems of the present invention. Accordingly, reductases, particularlythose capable of receiving FMN and nicotinic acids, may be utilized asthe oxidant component of the methods, compositions, and systems of thepresent invention. In addition, a number of other suitable oxidants willbe readily determinable by those skilled in the art.

Preferred oxidant compounds exhibit a one-electron reduction potential(E₀) between about −400 mV and about −165 mV inclusive, more preferablybetween about −396 mV and about −240 mV. Multiple electron reductionsare biologically important with P450 and oxygen. Examples of suitableoxidants include, but are not limited to, ferredoxin-NADP+ reductasesand NADPH:cytochrome P450 reductase including the reductases listedhereinabove, as well as flavins, nucleotides, nitrobenzoates, nicotinicacids, nitrobenzoic acids, ammonium salts, menadione, amine oxides,formamidines, cytochrome reductases, and slow-release formularies,salts, hydrates, aldehydes, esters, amines, amides, alcohols,derivatives, dietary supplements and other biologically or chemicallyequivalent derivatives thereof and combinations thereof.

Specific examples of flavins which are useful as oxidants in the methodsand compositions of the present invention include, but are not limitedto, riboflavin, flavin mononucleotide (FMN), menadione, deazaflavin,lumichrome, lumizine, flavin adenine dinucleotide (FAD), alloxazine,salts, hydrates, derivatives and combinations thereof. Specific examplesof ammonium compounds include, but are not limited to ammonium sulfate,ammonium chloride, ammonium hydroxide, urea, amines, and the like.Specific examples of cytochrome reductases include, but are not limitedto, cytochrome f, Cc, cytochrome b5, flavocytochrome P450, nitric oxidesynthase, and combinations thereof. Specific examples of nicotinic acidsinclude but are not limited to NicoSpan, NiaSpan, niacin, NAD, NADH,NADP, NADPH and combinations thereof. Examples of preferred oxidants,whose selection is based on E₀ (values) and beneficial metabolism,include nitrobenzoate (−396 mV), FMN (−313 mV), glycolate (α) (−290 mV),riboflavin (−292 mV), and salts, hydrates and derivatives of any of theabove.

Currently preferred oxidants for use in the methods and compositions ofthe present invention include but are not limited to FMN, NiaSpan,NAD/NADH, menadione, ammonium sulfate, α, and salts, hydrates andderivatives thereof FMN and nicotinic acids are particularly preferredoxidants in the compositions, methods and systems of the presentinvention, primarily because they are cost effective and generallyregarded as safe. Additionally, organ products such as whole liver, dryliver pills, liver oils and liver extracts are excellent sources ofundefined cytochromes, oxidants, and reductants. Where organ productsare unpalatable or are problematic because of, for instance, cholesterolor gout, it would be advisable to avoid these types of products.Moreover, in the course of processing, proteins and nucleotides may bedenatured; thus, organ-derived products may require supplementation withoxidants. FMN is the particularly preferred oxidant.

As noted above, oxidants are employed solely or, they may comprise anyone or more of the foregoing oxidants in combination with a reductant orother oxidants. For example, in one preferred embodiment, the oxidantcomprises a combination of FMN and fish oil concentrate. In theembodiment of the invention wherein two or more oxidants are combined,they are typically equimolar provisions of the oxidant component of thecompositions and methods of the present invention.

Inhibitors

A partial list of inhibitors includes the following common items: foodsand beverages, including grapefruit, turmeric (curcuminoids),bergamottin (flavoring in Earl Grey Tea), and well-water (arsenic);drugs, such as, antibiotics (ciprofloxacin, erythromycin,chloramphenicol, and the like), antivirals (ritonavir, etc.);contraceptives (ethynylprogesterone, and the like), antimycotics(ketoconazole, itraconazole, terconazole, miconazole, and the like),chemotherapies (aminoglutethimide, etc.); acid reflux (cimetidine),bioassays (metyrapone, diethyldithiocarbamate), barbiturates and mooddrugs (secobarbital, Prozac, Zoloft, Luvox and the like); cosmetics(hydrogen peroxide, dental whitener, bleach, etc.); air pollution fromcarbon monoxide and other components of smog; insecticides (cyanides,parathion, piperonyl butoxide, etc.); and acyl hydrazine, alkylhydrazine, aryl hydrazine, allylisopropylacetamide, carbon disulfide,carbon tetrachloride, dichloroacetamides, dihydropyridine, disulfiram,isothiocyanate, mercaptosteroid, phenylimidazole, phenelzine,phenylphenanthridinone, quinolones, syndones, thiourea, tienilic acid,and undecynoic acid.

In a physiological sense, constant exposure to inhibitors renders thegeneral population susceptible to xenobiotics. When P450 is inhibited,it is imperative to resurrect dysfunctional P45:FMN complexes by thecompositions and methods of the present invention, if for no otherreason than to reduce susceptibility to carcinogens. Inhibitors includecompounds that degrade or bind to the heme iron atom or to theprosthetic heme group. Others may be competitive inhibitors of P450, theeffects of which may last the half life of a drug.

Application

The oxidants are, by themselves useful in methods of treatment and inmethods of fortifying the P40 enzyme complexes. For example, flavins, bythemselves, or together with suitable excipients and/or carriers, areuseful in the methods of the present invention. Although the oxidantcomponents may be applied in a solid form, it is sometimes advantageousto provide oxidants in liquid form, such as by solubilizing thecomponent in an aqueous or suitable organic solvent or carrier toproduce aqueous or organic solutions of the oxidants. The amount ofoxidant that is solubilized in the carrier will depend upon theparticular oxidant selected and the method of application. The oxidantmay be solubilized in the carrier by adding the oxidant to the carrierand allowing the oxidant to dissolve. In some instances, the applicationof stirring, agitation, or even heat may facilitate the dissolution ofthe oxidant in the carrier.

For example, suitable formulations are not particularly limited, andinclude solutions of the oxidant dissolved in a suitable carrier. Forexample, FMN can be dissolved in a carbonated beverage with or without areducing sugar such as xylose and/or glucose. The carbon dioxideinhibits premature oxidation of the FMN. FMN in combination withchocolate is another example, such as a formulation comprising 0.2 to0.5 mg FMN per 30-60 grams of chocolate, preferably dark chocolate. Yetother suitable formulations include the oxidant and one or more reducingsugars, artificial or natural, as discussed above. Still furthersuitable formulations include emulsions, such as FMN and fish oil, codliver oil or both. These emulsions can be formulated in a capsule andadministered orally. Another suitable formulation is FMN and red wine.

The oxidant can be administered in a carrier to create a formulationhaving an oxidant concentration in the range between about 0.0001% andabout 100% by weight of the composition inclusive, preferably betweenabout 0.01% and about 100% inclusive. For example, because ethanol is aninducer of CYP2E1, a flavin:alcohol beverage ratio of 1:1000 ispreferred to match ratios generally found to induce the enzyme complex.An exemplary skin cream comprising 0.1% to 1% α-hydroxy is suitable fortopical application when formulated with 0.001% to 0.005% riboflavin.

Compositions of the present invention may also include any of a varietyof excipients, which improve or at least do not hinder the beneficialeffects of the compositions of the present invention. While thecompositions of the present invention may consist essentially ofoxidants, compounds may be formulated in suitable excipients. In theembodiment wherein the oxidant is a single composition for use in themethods of the present invention, the composition may include excipientshaving solubilized, dispersed, supported, or otherwise containedtherein, an amount of the oxidant that induces cytochrome P450. Asolution containing oxidants may be prepared using the generaltechniques set forth above.

Compositions containing oxidants in a single solution may include anycombination of oxidants selected from those described hereinabove.Preferred oxidants for one-step compositions include, but are notlimited to FMN and nicotinic acids. For example, one compositionaccording to the present invention includes menadione and niacin.Another composition according to the present invention includes FMN andcod liver oil. Another composition according to the present inventionincludes daily 50 mg riboflavin and 5 mg NADP. Another compositionaccording to the present invention includes 1 mg FMN cloaked in carbondioxide as a stabilizing excipient and to be taken up to 5 times daily.

Compositions of oxidants will typically be administered at aconcentration ranging between about 1 mg and about 1000 mg per dose.Preferred combined oxidant compositions include: (1) 5 mg NADH and 25 mgniacin; (2) 5 mg FMN and 50 mg slow-release niacin; and (3) 3 mg FMN and500 mg fish oil concentrate. Macromolecules such as CPR and CYP poseproblems of stability and may require cold storage and inertenvironments. Formulation with carbonates or bicarbonates preventsoxidation. The compositions of the present invention may also be appliedtopically in appropriate dermatological or optical formulations.

Compositions according to the present invention may find specificallytailored utilization, including enhanced performance of NADPH:cytochromeP450 reductase; enhanced quantity of NADPH:cytochrome P450 reductase;fortification of P450:FMN enzyme complexes; inducing such complexesafter exposure to inhibitors; reduced susceptibility to carcinogens; andfor therapy. Compositions may also be formulated at very lowconcentrations of FMN or in sustained release dosages of niacin fordaily enhancement.

The aqueous solutions employed in the systems of the present inventionmay be formulated in the same manner as described hereinabove forcompositions, using the same types of aqueous carriers. Preferably apharmaceutically acceptable carrier is used. One preferred formulationaccording to the present invention includes flavin mononucleotide as theoxidant. Another preferred formulation according to the presentinvention includes niacin as the oxidant. Another preferred systemaccording to the present invention includes flavin mononucleotide andliver extracts as oxidants. Another formulation according to the presentinvention includes oxidants formulated with reductants such as, reducingsugars; the preferred formulation utilizing treatment with 1 mg to 100mg NiaSpan, sustained release niacin, as an oxidant formulated with 10 gto 1000 g of a pentose reductant such as xylose.

The following examples are provided to further illustrate the presentinvention, and should not be construed as limiting thereof. The presentinvention is defined by the claims, which follow.

In these examples, reagents, biochemicals, and dietary supplements wereobtained in purest form available, and were obtained as reagent gradeand USP chemicals where possible. Purified P450s including recombinanthuman NADPH:Cytochrome P450 Reductase (hCPR) were obtained from PanVera.

In these examples, “L” means liter; “ml” means milliliter; “cm” meanscentimeter; “cm² means centimeters squared; “nm” means nanometer; “M”means molar; “mM” means millimolar; “μM” means micromolar; “nM” meansnanomolar; “mol” means moles; “μmol” means micromoles; “mg/ml” meansmilligrams per milliliter; “ml/cm²” means milliliters per centimetersquared; “kDa” means kiloDaltons; “L/min” means liters per minute; “d”means days; “h” means hours; “min” means minutes; “s” means seconds; “g”means multiple of centrifugal gravitational force; “°” means degreescentigrade; CYP is a cytochrome P450 monooxygenase; CPR isNADPH:cytochrome P450 reductase; CPR is microsomal-CPR.

The following exemplary compositions are intended to provide furtherguidance to those skilled in the art, and do not represent an exhaustivelisting of compositions within the scope of the present invention.

Example I First Exemplary Composition: FMN

Concentration per dose Composition Broad Range Narrow Range FMN 0.01-50mg 3-5 mg Cod liver oil 1-2500 mg 100-1000 mg

Second Exemplary Composition: Drink

Concentration per 100 ml dose Composition Broad Range Narrow RangeXylose 0.1-1% 1-1000 g FMN 1-1000 ppm 1 mcg-5 mg

Third Exemplary Composition

Concentration Component Broad Range Narrow Range FMN 0.01-20 mg 1-5 mgAnalgesics 1-2000 mg 5-100 mg

Fourth Exemplary Composition

Concentration Composition Broad Range Narrow Range FMN 1-5 mg 2-5 mgNADP 10-100 mg 10-50 mg Bicarbonate excipient excipient

Fifth Exemplary Composition

Component Dose Range FMN 5 mg 1-5 mg Liver extract 1 mg 10 mcg-2 g

Sixth Exemplary Composition: Drink

Concentration Composition Broad Range Narrow Range FMN 1 mcg-50 mg 10mcg-5 mg D-Xylose 1-1000 g 5-50 g EtOH excipient excipient Carbondioxide excipient excipient Water excipient excipient

Seventh Exemplary Composition: Natural Sweetener Suitable for Peoplewith Diabetes

Concentration Composition Broad Range Narrow Range Folate 1-100 mcg 1-3mcg FMN 1-8000 mcg 1-100 mcg D-Xylose 1-1000 g 5-50 g

Eighth Exemplary Composition: Blended Diet Sweetener

Concentration Composition Broad Range Narrow Range FMN 1-1000 mcg 2-200mcg D-Xylose 1-1000 g 5-50 g Sucralose 1-1000 g 5-50 g

Example 2

The following example illustrates an application of human P450compositions according to the present invention. Procedures followedpreviously described methods, e.g., U.S. Pat. No. 6,020,288; M.Markwell, et al., Methods of Enzymology 72:296-303 (1981); C. A.Mihaliak, et al., Methods in plant biochemistry 19:261-279 (1993); R.Donaldson, et al., Arch. Biochem. Biophys. 152:199-215 (1972); and M.Persans, et al., Plant Physiol. 109:1483-1490 (1995)). The resultsdemonstrate the efficacy of the methods and compositions of the presentinvention for the enhancement of P450 enzyme complexes.

Methods. CPR substrates have been assayed on various tissues (e.g., U.S.Pat. No. 6,020,288); therefore, response to human CPR (hCPR) wasexamined under controlled conditions on live index organisms. The directeffects of substrates on hCPR were measured by preparation of microsomesfor quantification against CPR and Cc. Controls included equalconcentrations of each individual treatment or substrate in surfactantand water. Untreated controls were maintained under identical conditionsof culture. Oxidase activity of Cc was inhibited by potassium cyanide.The reaction was initiated by addition of Cc wherein NADPH-dependentreduction of Cc was monitored for increases in absorbance.

Results and Discussion. FMN showed over 4.5 times the induction ofcomponents tested. Cytochromes P450 enzyme complexes have definedcatalytic electron transfer functions. See, C. von Wachenfeldt, et al.,Structures of Eukaryotic Cytochrome P450 Enzymes, P. R. Ortiz deMontellano, ed. (1995) Cytochrome P450: Structure, Mechanism, andBiochemistry (Second Ed.), Plenum Press, New York, pp 183-223 and H.Strobel, et al., NADPH Cytochrome P450 Reductase and Its Structural andFunctional Domains, P. R. Ortiz de Montellano, ed. (1995) CytochromeP450: Structure, Mechanism, and Biochemistry (Second Ed.) Plenum Press,New York, pp 225-244. Combinations of cytochromes P450 are numerous andunderscore the potential of the field. Selection of oxidants based onone electron reduction of compounds (see, e.g., Wardman, P. 1989, JPhys. Chem. Ref Data 18(4):1637-1755) within potentials associated withCPR reductase (see, e.g., Butler, J. et al. 1993 Biochimica etBiophysica Acta 1161:73) proved successful, test results showing thatspecific activity may be enhanced by FMN. Oxidants accelerate cytochromeP450 and, FMN, in particular, may fortify its integral structure. Insome instances, enhancement of cytochromes other than CPR may be key.For example, Cc may accelerate response where CPR does not, and forthose exceptional cases, broad spectrum dosage or specific agents willbe the subject of further investigations. Even though the most potenttreatment may be nanomolar cytochromes, of the integral components ofcytochromes, FMN showed activity at sufficiently low concentrations tobe a prime selection for practical, safe and effective therapies. P450provides widespread applicability of compositions and methods forselection of components which may be utilized to endow animals with ameans of resistance to xenobiotic stresses while gaining ever greaterhealth.

What is claimed is:
 1. A method of inducing P450:FMN complexes in ananimal, comprising: formulating a composition comprising a stabilizingexcipient, 1 mcg to 50 mg of FMN or a salt thereof protected by saidstabilizing excipient, one or more flavins selected from the groupconsisting of flavin adenine dinucleotide, lumichrome, lumizine,alloxazine and combinations thereof, and salts or hydrates thereof; anda member selected from the group consisting of a nicotinic acid, analpha hydroxy acid, an alcohol, and riboflavin; and an ammonium compoundselected from the group consisting of ammonium sulfate, ammoniumchloride, ammonium hydroxide and urea; and administering to said animalsaid composition in an amount effective to induce P450:FMN complexes insaid animal.
 2. The method of claim 1, wherein said animal is a human.3. The method of claim 1, wherein said composition further comprises asweetener.
 4. The method of claim 3, wherein said sweetener is selectedfrom the group consisting of glucose, fructose, xylose, sucrose,artificial sweetener, xylitol, disaccharide, oligosaccharide andcombinations thereof.
 5. The method of claim 1, wherein said compositionis in the form of a microencapsulated system or a cream.
 6. The methodof claim 1, wherein said composition is administered to said animaltransdermally or topically.
 7. The method of claim 1, wherein saidcomposition is in the form of a dietary supplement or a cosmetic.
 8. Themethod of claim 1, wherein said alpha hydroxy acid is in the form of itsester, and is a glycolate.
 9. The method of claim 1, wherein saidcomposition is in liquid form.
 10. The method of claim 1, wherein saidalpha hydroxy acid is present in an amount of 0.1 to 1%.
 11. The methodof claim 1, wherein said composition further comprises a fish oilselected from fish oil concentrates and cod liver oils.
 12. A method ofinducing P450:FMN complexes in an animal, comprising: formulating acomposition comprising a stabilizing excipient, 1 mcg to 50 mg of one ormore flavins selected from the group consisting of flavinmononucleotide, flavin adenine dinucleotide, lumichrome, lumizine,alloxazines and combinations thereof, and salts or hydrates thereof; analpha hydroxy acid; and a member selected from the group consisting of anicotinic acid, riboflavin, and an alcohol; and an ammonium compoundselected from the group consisting of ammonium sulfate, ammoniumchloride, ammonium hydroxide and urea; and administering to said animalsaid composition in an amount effective to induce P450:FMN complexes insaid animal.
 13. The method of claim 12, wherein said animal is a human.14. The method of claim 12, wherein said composition comprises one ormore alcohols.
 15. The method of claim 12, wherein said composition isin the form of a microencapsulated system or a cream.
 16. The method ofclaim 12, wherein said composition is administered to said animaltransdermally or topically.
 17. The method of claim 12, wherein saidcomposition is in the form of a dietary supplement or a cosmetic. 18.The method of claim 12, wherein said alpha hydroxy acid is in the formof its ester, and is a glycolate.
 19. The method of claim 12, whereinsaid composition further comprises alcohols selected from ethanol,alcohol beverage and red wine.